SI9110782A - 1,3-oxathiolane nucleoside analogues - Google Patents

Abstract

(-) - 4-amino-1- (2-hydroxymethyl-1,3- oxathio-lan-5-yl) - (1H) -pyrimidin-2-one, its pharmaceutical acceptable derivatives, its pharmaceuticals formulations, procedures for its preparation and its use as an antiviral agent.

Description

The present invention relates to nucleoside analogues and their use in medicine. More specifically, the invention relates to 1,3-oxathiolane nucleotioside analogues, to their pharmaceutical formulations and to their use in the treatment of viral infections.

A compound of formula (I) is described to have:

Also known as BCH-189 or NGPB-21 antiviral activity, specifically against human immunodeficiency viruses (HIV), agents that cause AIDS (5th AntiAids Conference, Montreal, Canada June 5-9, 1989: Abstracts TC0.1 and MCP 63 ; European Patent Application Publication No. 0382562). The compound of formula (I) is a racemic mixture of two enantiomers of formulas (1-1) and (1-2):

S and described and tested in the form of its racemate. The only compound currently accepted for treating HIV-induced conditions is 3′-azido-31-deoxythymidine (AZT, zidovudine, BW 5090). However, this compound has a significant tendency for side effects and therefore either cannot be used or should, once used, be withdrawn from therapy in a significant number of patients. As a result, there is a continuing need to provide compounds that are effective against HIV or have a significantly better therapeutic index.

We have now unexpectedly found that the enantiomers of a compound of formula (I) are equally potent against HIV, but that one enantiomer, (-) enantiomer, has significantly lower cytotoxicity than the other enantiomer. Thus, in the first aspect of the invention, the (-) (or left-handed) enantiomer of a compound of formula (I) and its pharmaceutically acceptable derivatives are provided.

The (-) enantiomer has the chemical name (-) - 4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) (1H) -pyrimidin-2-one (hereinafter referred to as compound (A )). It has the absolute stereochemistry of a compound of formula (1-1) called (2R, cis)) - 4-amino-1- (2hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2- he.

Preferably, compound A is provided substantially free of the corresponding (+) - enantiomer, i.e., no more than about 5% w / w (+) - enantiomer is present, preferably not more than about 2%, especially less than about 1% w / w m.

A pharmaceutically acceptable derivative is understood to mean any pharmaceutically acceptable salt, ester or salt of such ester of compound (A) or any other compound which, upon administration to a patient, may provide (direct or indirect) compound (A) or an antiviral active metabolite or residue thereof.

It will be appreciated by those skilled in the art that compound (A) can be modified on the functional groups in both the basic moiety and the hydroxymethyl group of the oxathiolane ring to provide its pharmaceutically acceptable derivatives. Modifications of all such functional groups are included within the scope of the invention. However, the pharmaceutically acceptable derivatives obtained by the modification of the 2-hydroxymethyl group of the oxathiolane ring are of particular interest.

Preferred esters of compound (A) include compounds in which the hydrogen of the 2-hydroxymethyl group is replaced by an acyl function R-C = O in which the non-carbonyl portion of the R ester is

selected from hydrogen, straight or branched chain alkyl groups (e.g. methyl, ethyl, n-propyl, t-butyl), alkoxyalkyl (e.g. methoxymethyl), aralkyl (e.g. benzyl), aryloxyalkyl (e.g. phenoxymethyl), aryl (e.g. eg. phenyl, which is optionally substituted by halogen, C ₇ ^ alkyl or _C ^ alkoxy); sulfonate esters such as alkyl or aralkylsulfonyl (e.g. methanesulfonyl); amino acid esters (e.g., L-valyl or L-isoleucyl) and mono, di- or tri-phosphate esters.

According to the esters described above, unless otherwise indicated, any alkyl group present contains suitably 1 to 16 carbon atoms, in particular 1 to 4 carbon atoms. Any aryl group present in such esters appropriately comprises a phenyl group.

The esters may in particular be C _{7 76} alkyl ester, unsubstituted benzyl ester or benzyl ester substituted with at least one halogen (bromine, chlorine, fluorine or iodine),

C _1-6 alkyl, C _; alkoxy, nitro or trifluoromethyl groups.

The pharmaceutically acceptable salts of compound (A) include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acids include hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-psulfonic, tartaric, acetic, citric, methanesulfonic, formic, benzoic, malonic, malonic, 2- sulfonic and benzenesulfonic acid. Other acids, such as oxalic, which by themselves are not pharmaceutically acceptable, may be useful as intermediates for the preparation of the compounds of the invention and their pharmaceutically acceptable acid addition salts.

Salts derived from appropriate bases include alkali metal (eg. Sodium), alkaline earth (eg. Magnesium), ammonium and NR ₄ ⁺ (where R _c; alkyl).

Subsequent references to the compound of the invention include both compound (A) and pharmaceutically acceptable derivatives thereof.

The compounds of the invention possess antiviral activity either per se or may be metabolized to such compounds. In particular, these compounds are effective in inhibiting the replication of retroviruses, including human retroviruses, such as human immunodeficiency virus (HIV), agents that cause AIDS.

Thus, as a further aspect of the invention, compound (A), or a pharmaceutical, is provided

an acceptable derivative for use as an active therapeutic agent, especially as an antiviral agent, e.g. for the treatment of retrovirus infections.

In a further alternative aspect, there is provided a method for treating a viral infection, in particular an infection caused by such a retrovirus such as HIV in a mammal, including a human, comprising administering an effective amount of compound (A) or a pharmaceutically acceptable derivative thereof.

In another alternative aspect, the use of compound (A) or a pharmaceutically acceptable derivative thereof for the manufacture of a medicament for the treatment of a viral infection is also ensured.

The compounds of the invention are also useful in the treatment of conditions related to AIDS, such as the AIDS-related complex (ARC), progressive generalized lymphadenopathy (PGL), AIDS-like neurological diseases (such as dementia or tropical paraparesis) , anti-HIV antibodies positive and HlV-positive conditions, Kaposi's sarcoma, thrombocytopenia of purpurea and accompanying opportunistic infections, e.g. Pneumocystitis carinil.

The compounds of the invention are also useful in preventing the progression of clinical disease in individuals who are anti-HIV antibodies or HlV-antigen positive and in prophylaxis after HIV exposure.

Compounds (A) or their pharmaceutically acceptable derivatives can also be used to prevent viral contamination of saline fluids such as blood or semen in vitro.

It will be appreciated by those skilled in the art that the indication of treatment herein refers to both the prophylaxis and the treatment of established infections or symptoms.

Furthermore, it will be appreciated that the amount of a compound of the invention required for use in treatment will not only vary with the selected compound, but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient. will be determined by the attending physician or veterinarian. However, a suitable dose will generally be in the range of about 0.1 to about 750 mg / kg body weight per day, preferably in the range of about 0.5 to about 60 mg / kg / day, most preferably in the range of about 1 to about 20 mg / kg / day.

The desired dose may be suitably formulated into a single dose or into divided doses administered at appropriate intervals, e.g. as two, three, four or more sub-doses per day.

The compound is suitably administered in a unit dosage form, e.g., containing 10 to 1500 mg, suitably 20 to 1000 mg, most preferably 50 to 700 mg of the active ingredient per unit dosage form.

Ideally, the active ingredient should be administered in such a way that a maximum concentration of the active ingredient in the plasma is obtained from about 1 to about 75 μΜ, preferably about 2 to 50 μΜ, most preferably about 3 to about 30 μΜ. This can be achieved, e.g. by intravenous injection of a 0.1 to 5% solution of the active ingredient, optionally in saline or by oral administration of a bolus containing about 1 to about 100 mg of the active ingredient. The desired blood levels can be maintained by continuous infusion by providing about 0.01 to about 5.0 mg / kg / hour or intermittent infusions containing about 0.4 to about 15 mg / kg / active ingredient.

Although it is possible to administer the compound of the invention as a crude chemical for use in therapy, it is preferable to formulate the active ingredient as a pharmaceutical formulation.

The invention further provides a pharmaceutical formulation comprising compound (A) or a pharmaceutically acceptable derivative thereof together with one or more pharmaceutically acceptable carriers therefor and, optionally, other therapeutic and / or prophylactic ingredients. The carrier (s) must be acceptable in the sense that they are compatible with the other ingredients of the formulation and are not harmful to the patient.

Pharmaceutical formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration in a form suitable for administration by inhalation or insufflation. The formulations may, where necessary, be suitably formulated into separate dosage units and may be prepared by any of the methods well known in the pharmaceutical art. All processes involve the step of bringing the active ingredient into contact with the liquid carriers or the finely divided solid carriers or both, and then, if necessary, forming the product into the desired formulation.

Pharmaceutical formulations suitable for oral administration may be formulated as separate units, such as capsules, bags or tablets, each containing a predetermined amount of the active ingredient: as a powder or granules; as a solution, as a suspension or as an emulsion. The active ingredient can also be formulated as a bolus, pulp or paste. Tablets and capsules for oral administration may contain such conventional ingredients as binders, fillers, lubricants, disintegrants or wetting agents. The tablets may be coated according to techniques well known in the art. Oral liquid preparations may take the form of e.g. aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be formulated as a dry product for constitution with water or other suitable carrier prior to use. Such liquid preparations may contain such conventional additives as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils) or preservatives.

The compounds of the invention can also be formulated for parenteral administration (eg by injection, e.g., bolus injection or continuous infusion), and can be formulated into a single dosage form into ampoules, pre-filled injections, low volume infusion, or multiple dosage containers with added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles and may contain formulating agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active form may be in the form of a powder obtained by aseptic isolation of a sterile solid or by lyophilization from solution, for constitution with some suitable carrier, e.g. with pyrogen-free sterile water, before use. For topical application to the skin, the compounds of the invention may be formulated as ointments, creams or lotions, or as a transdermal patch. Ointments and creams may e.g. formulate with an aqueous or oily base by adding suitable thickeners and / or gels. Lotions can be formulated with a water or oil base and will also generally contain one or more emulsifying, stabilizing, dispersing, suspending, thickening or coloring agents.

Formulations that are suitable for topical administration to the mouth include lozenges that comprise the active ingredient in a fragrant base, usually sucrose and acacia or tragacanth; a lozenge containing an active ingredient on such an inert basis as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier. Pharmaceutical formulations suitable for rectal administration in which the carrier is a solid are most preferably formulated as suppositories in the form of dosage units. Suitable carriers include cocoa butter and other materials commonly used in the art, and suppositories may

is suitably molded by mixing the compound with a softened or molten support (i) followed by strong cooling and molding.

Formulations suitable for vaginal administration may be formulated as pessaries, tampons, creams, gels, pastes, foams or sprays containing, in addition to the active ingredient, such carriers as are known in the art to be suitable.

For intranasal administration, the compounds of the invention can be used as a liquid spray or dispersible powder or in droplets.

The droplets may be formulated with an aqueous or non-aqueous base which also contains one or more dispersing agents, dissolving agents or suspending agents. Liquid sprays are received from pressurized packaging.

For administration by inhalation, the compounds of the invention are administered conveniently from insufflators, nebulizers or pressurized packs or other suitable aerosol dispenser. Pressure packs may include such suitable propellant as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve to deliver the metered amount.

Alternatively, the administration of the compounds of the invention may be effected by inhalation or insufflation in the form of a dry powder preparation, e.g. mixtures of powder compounds and some suitable powder bases such as lactose or starch. The powder preparation can be formulated in a unit dosage form in e.g. capsules or cartridges or e.g. in gelatin or blister packs from which the powder may be administered by means of an inhaler or insufflator.

When desired, the formulations described above are adapted so that they can be used to give the active ingredient in sustained release form.

The pharmaceutical compositions of the invention may also contain other active ingredients such as antimicrobial agents or preservatives.

The compounds of the invention may also be used in combination with other therapeutic agents, e.g. with other anti-infectious agents. In particular, the compounds of the invention may be used in conjunction with known antiviral agents.

The invention thus provides, in a further aspect, a combination comprising compound (A) or a physiologically acceptable derivative thereof together with another therapeutically active agent, in particular an antiviral agent.

The combinations discussed above can be suitably formulated for use in the form of a pharmaceutical formulation, and such pharmaceutical formulations comprising the combination as defined above together with a pharmaceutically acceptable carrier form a further aspect of the invention.

Suitable therapeutic agents for use in such combinations include acyclic nucleosides such as acyclovir or ganciclovir, interferons such as α, β or 7-interferon, renal excretion inhibitors such as probenecid, nucleoside transport inhibitors such as dipyridamole, 2 ', 3 '-dideoxynucleosides such as AZT, 2', 3'-dideoxycytidin-2 ', 3'-dideoxyadenosine, 2', 3'-dideoxythymidine, 2 ', 3'-dideoxy-2', 3'-didehydrothymidine and 2 ' , 3'-dideoxy-2 ', 3'-didehydrocytidine, immunomodulators such as interleukin II (IL2) and granulocytic macrophage colony stimulating factor (GM-CSF), erythropoietin, ampligen, thymomodulin, thymopentin, foscarnet, ribavirin and binding inhibitors HIV to CD4 receptors, e.g. soluble CD4, CD4 fragments, CD4 hybrid molecules, glycosylation inhibitors such as 2-deoxy-D-glucose, castanospermine and

1-deoxynoirimycin.

The individual components of such combinations may be administered either sequentially or simultaneously in special or combined pharmaceutical formulations.

When compound (A) or a pharmaceutically acceptable derivative thereof is administered in combination with another therapeutic agent that is active against the same virus, the dose of each compound may be the same or different from that used when the compound is used alone. Those skilled in the art will readily receive the appropriate dosage.

Compound (A) and its pharmaceutically acceptable derivatives can be prepared by methods known in the art for the preparation of compounds of analogous structure, e.g. as described in European Patent Application Publication no. 0382562.

It will be appreciated by those skilled in the art that for the particular methods described below, the desired stereochemistry of compound (A) can be obtained either by starting from an optically pure starting material or by decomposing the racemic mixture at any suitable synthesis stage. In all procedures, the optically pure desired product can be obtained by repeating 0-1 R .M - 88 ,. j y% decomposition of the final product of each reaction.

In such process (A) 1,3-oxathiolane of formula (VIII):

in which the anomeric group L is a replaceable group is reacted with a suitable base. Suitable L groups include -OR, where R is an allyl group, e.g.

A C _1-6 alkyl group such as methyl or R is an acyl group, e.g. A C _1-6 alkyl group such as acetyl or halogen, e.g. iodine, bromine or chlorine.

The compound of formula (VIII) is suitably reacted with cytosine or a suitable pyrimidine base precursor (previously silylated by such a silylating agent such as hexamethyldisilazane) in some such compatible solvent as methylene chloride, using some such Lewis acid as titanium tetrachloride, a tin (IV) compound such as SnCl ₄ or trimethylsilyl triflate.

The 1,3-oxathiolanes of formula (VIII) can be prepared e.g. by reacting an aldehyde of formula (VII) with a mercaptoacetal of formula (VI) in some such compatible organic solvent as toluene in the presence of an acid catalyst, e.g. some such Lewis acids as zinc chloride.

HSCH ₂ CH (OC ₂ H ₅ ) ₅ (VI)

C ₆ H ₅ CO ₂ CH ₂ CHO (VII)

The mercaptoacetals of formula (VI) can be prepared by methods known in the art, e.g. G. Hesse and I. Jorder, Chem. Ber. 85, 924-932 (1952).

Aldehydes of formula (VII) can be prepared by known methods in the art, e.g. Е.

G. Halloquist and H. Hibbert, Can. J. Research, 8, 129-136 (1933). Suitably, the crude aldehyde (VII) can be purified by conversion to the crystalline bisulfite addition adduct and subsequent conversion to the free aldehyde.

In the second process (B), compound (A) is obtained by internal conversion using the base of the compound of formula (IX):

B

HOCH

C1X9 where B is a base that can be converted to cytosine by the session. Such internal conversion can proceed either by simple chemical transformation (eg by conversion of uracil base to cytosine) or by enzymatic conversion using deoxyribosyl transferase. Such processes and conditions for internal base conversion are well known in the art of nucleoside chemistry.

In the third process (C), a compound of formula (XI) can:

nh ₂

CXI9 is converted to compound (A) by conversion of the NH ₂ group to a cytosine base by well-known methods in the nucleoside chemistry technique.

Many of the reactions described later are extensively published in the context of nucleoside synthesis, e.g. in Nucleoside Analogs - Chemistry, Biology and Medical Applications, by R. T. Walker et. al., ed. Plenum Press, New York (1979) at p. 165-192; T. Ueds in Chemistry of Nucleosides and Nucleotides, vol. I, L. B. Townsend ed., Plenum Press, New York (1988) at p. 165-192, the descriptions of which are incorporated herein by reference.

It will be appreciated that the above reactions may require, or may be commonly used on them, starting materials having protected functional groups and thus removing the protection as an intermediate or final step to obtain the desired compound. Protection and deprotection of functional groups can be carried out using conventional means. Thus, e.g. the amino groups are protected with a group selected from aralkyl (e.g. benzyl), acyl, aryl (e.g. 2,4-dinitrophenyl) or silyl; subsequent removal of the protecting group is carried out, where desired, by hydrolysis or hydrogenolysis, as appropriate, using standard conditions. Hydroxyl groups can be protected using a conventional hydroxyl protecting group, e.g. as described in Protective Groups in Organic Chemistry, ed. J.F.W. McOmie (Plenum Press, 1973) or Theodor W.Greene's Protective Groups in Organic Synthesis (John Wiley and Sons, 1981). Examples of suitable hydroxyl protecting groups include groups selected from alkyl (e.g. methyl, t-butyl, or methoxymethyl), aralkyl (e.g.

/ A

benzyl, diphenylmethyl or triphenylmethyl), heterocyclic groups such as tetrahydropyranyl, acyl (e.g. acetyl or benzoyl) and silyl groups such as trialkylsilyl (e.g. t-butyldimethylsilyl). Hydroxyl protecting groups can be removed by conventional techniques. Thus, e.g. alkyl, silyl, acyl and heterocyclic groups are removed by solvolysis, e.g. hydrolysis under acidic or basic conditions. Aralkyl groups such as triphenylmethyl can be removed in a similar manner by solvolysis, e.g. hydrolysis under acidic conditions. Aralkyl groups such as benzyl can be cleaved, e.g. by treatment with BF ^ / ether and acetate anhydride and then separating the resulting acetate groups at the appropriate synthesis stage. The silyl groups can also be conveniently removed using a source of fluoride ions, such as tetra-n-butyl ammonium fluoride.

In the above procedures, compound (A) is generally obtained as a mixture of cis and trans isomers, of which the cis isomer is an interesting compound.

These isomers can be separated by physical means, e.g. by chromatography on silica gel or by fractionation crystallization or directly or on their corresponding derivatives, e.g. acetates (prepared eg with acetic anhydride) and then, after separation, re-converted to the original product (eg by deacetylation with methanolic ammonia).

The pharmaceutically acceptable salts of the compounds of the invention can be prepared as described in U.S. Pat. No. 4,383,114, the description of which is incorporated herein by reference. Thus, for example, when the acid addition salts of compound (A) are desired, the product of any of the above processes can be converted into a salt by conventional treatments using a suitable free base with some suitable acid. Pharmaceutically acceptable acid addition salts may be prepared by reaction of the free base with the appropriate acid, optionally in the presence of some suitable solvent such as an ester (eg ethyl acetate) or an alcohol (eg methanol, ethanol or isopropanol). Salts with an inorganic base may be prepared by reaction of a base compound with some such suitable base as an alkoxide (e.g. sodium methoxide), optionally in the presence of some such solvent as an alcohol (e.g. methanol). Pharmaceutically acceptable salts may also be prepared from other salts, including the pharmaceutically acceptable salts of compound (A) using conventional procedures.

Compound (A) can be converted to a pharmaceutically acceptable ester or other ester by reaction with a phosphorylating agent such as POCl ₅ or some suitable esterifying agent, such as an acid halide or anhydride, if necessary. The ester or salt of compound (A) can be converted to the parent compound, e.g. by hydrolysis.

The decision of the final product or intermediate or starting material thereto can be made by any suitable method known in the art: see e.g. Stereochemistry of Carbon Compounds, by E. L. Eliel (McGraw Hill, 1962) and Tables of Resolving Agents, by S. H. Wilen.

Thus, e.g. compound (A) is obtained by chiral HPLC using a suitable stationary phase, e.g. acetylated / 3-cyclodextrin or cellulose triacetate and a suitable solvent, e.g. such alcohol as ethanol or aqueous solutions, e.g. of triethylammonium acetate. Alternatively, the compounds can be selected by enantioselective catabolism mediated by enzymes with some such suitable enzyme, such as cytidine deaminase or selective enzymatic degradation of some suitable derivative, using 5′-nucleotidase. When enzymatic decision-making is performed, the enzyme may be used either in solution or, more preferably, in immobilized form. The enzymes can be immobilized by any known method in the art, e.g. by adsorption on a resin such as Eupergit C.

The invention is further described by the following examples, which are not intended to limit the invention in any way. All temperatures are in degrees Celsius.

INTERMEDIAT1

5-methoxy-1,3-oxathiolan-2-methanol, benzoate

A solution of zinc chloride (1.6 g) in hot methanol (15 ml) was added to a mixed solution of mercaptoacetaldehyde, dimethylacetal (34.2 g) and benzoyloxyacetaldehyde (48.3 g) in toluene (1300 ml), which was then heated at reflux. under nitrogen for 50 minutes. The cooled mixture was concentrated, diluted with toluene, then filtered through silica. The combined filtrates and toluene were washed with saturated aqueous sodium carbonate solution (x2) and dried with brine (MgSOJ), then evaporated to an oil which was subjected to silica column chromatography (2 kg, Merck 9385), eluting with chloroform, thus to give the title product as an oil (45.1 g), a mixture of anomers (ca 1: 1) IH NMR (DMSO-d _fi), 3.1-3.3 (4H), 3.42 (6H), 4.4 -4.6 (4H), 5.41 (1H), 5.46 (1H), 5.54 (1H), 5.63 (1H), 7.46 (4H), 7.58 (2H) , 8.07 (4H); θ max (CHBrp 1717 cm ' ⁷ .

INTERMEDIATE 2 (±) -cis-1- (2-benzoyloxymethyl-L3-oxathiolan-5-yl) - (1H) -pyrimidine-2,4-dione

A mixture of finely ground uracil (9.62 g), hexamethyldisilazane (50 ml) and ammonium sulfate (30 mg) was refluxed under nitrogen until a clear solution was obtained. This was cooled and then evaporated to a colorless oil which was dissolved under an atmosphere of nitrogen in acetonitrile (100 ml). The solution was added to a stirred, ice-cooled solution of 5-methoxy-1,3-oxathiolan-2-methanol, benzoate (intermediate i) (19.43 g) in acetonitrile (600 ml) and trimethylsilyltrifluoromethanesulfonate (14.7 ml) was added. The ice bath was removed and the solution was heated at reflux under nitrogen for 45 minutes. After cooling and evaporation, the residue was purified by column chromatography over 1 kg of silica gel (Merck 9385), eluting with chloroform / methanol 9: 1. The appropriate fractions were combined and evaporated to give a crude residue. This fractionation was crystallized from minimally hot methanol (about 1200 ml) to give the title compound (6.32 g) as white crystals. 1 H NMR (d ⁶ DMSO) δ 11.36 (1H, broad s), 7.50-8.00 (6H, m), 6.20 (1H, t), 5.46 (2H, m). 4.62 (2H, m), 3.48 (1m, m), 3.25 (1H, m).

INTERMEDIATE 3 (±) - (cis-4-amino-1- (2-benzoyloxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one

Procedure (s)

A suspension of cytosine (20,705 g) and ammonium sulfate (several mg) in hexamethyldisilazane (110 ml) was stirred and refluxed for 2.5 hours under nitrogen. The solvent was removed by evaporation and the resulting solid was dissolved in dry acetonitrile (350 ml). Transfer this solution using flexible needle techniques to a mixed, ice-cooled solution of 5-methoxy-1,3-oxathiolan-2-methanol benzoate (intermediate I) (43.57 g) in acetonitrile (650 ml) under nitrogen. Trimethylsilyltrifluoromethanesulfonate (33 ml) was added, the solution was allowed to warm to room temperature (1.5 hours) and then refluxed overnight. The residue was concentrated, diluted with saturated aqueous sodium bicarbonate solution (500 ml), then extracted with ethyl acetate (3x500 ml). The combined extracts were washed with water (2x250 ml) and brine (250 ml), dried (MgSO ₄ ), then evaporated to a foam, which was chromatographed on a silica column (600 g, Merck 7734), eluted with ethyl acetate-methanol mixtures to give a mixture of anomers (about 1: 1,

31.59 g). The mixture was crystallized from water (45 ml) and ethanol (9.0 ml) to give a solid (10.23 g) which was recrystallized from ethanol (120 ml) and water (30 ml) to give the title product as white solid (9.26 g); kmax (MeOH) 229.4 mm (E ^7% 610); 272.4 mm (E ' ^% 293); 1 H NMR (DMSO d 6) δ 3.14 (1H), 3.50 (1H), 4.07 (2H), 5.52 (1H), 5.66 (1H), 6.28 (1H). 7.22 (2H), 7.56 (2H), 7.72 (2H), 8.10 (2H).

Procedure (b)

Phosphorus oxychloride (7.0 ml) was added dropwise to a stirred ice-cold suspension of 1,2,4-triazole (11.65 g) in acetonitrile (120 ml) and then added dropwise while maintaining the internal temperature below 15 ° C. triethylamine (22.7 ml). After 10 minutes, a solution of (±) -cis-1- (2-benzoyloxymethyl-1,3-oxathiolan-5-yl) (1H) -pyrimidine-2,4-dione (intermediate 2) (6.27 g) was slowly added. in acetonitrile (330 ml). Then continue stirring at room temperature overnight. The mixture was cooled overnight with an ice bath and slowly added triethylamine (30 ml) followed by water (21 ml). The resulting solution was evaporated and the residue partitioned between saturated sodium bicarbonate solution (400 ml) and chloroform (3x200 ml). The combined chloroform extracts were dried with magnesium sulfate, filtered and evaporated to give a crude residue (9.7 g). The residue was dissolved in 1,4-dioxane (240 ml) and added to a concentrated aqueous ammonia solution (s. 0.880 g, 50 ml). After 1.5 hours, the solution was evaporated and the residue dissolved in methanol. This causes precipitated solids to be filtered off. The mother liquor was purified by chromatography over a silica gel column (Merck 9385, 600 g). Combine the corresponding fractions to give the title compound as a tan solid (2.18 g) identical to that obtained by process (a).

Example 1 f) - (cis) -4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1-H) -pyrimidin-2-one

A suspension of (cis) -4-amino-1- (2-benzoyloxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin2-one (intermediate 3) (8.19 g) and Amberlite-IRA- 400 (OH) resins (8.24 g) in methanol (250 ml) were stirred and refluxed for 1.5 hours. The solids were separated by filtration and then washed with methanol. Evaporate the combined filtrates. The residue was triturated with ethyl acetate (80 ml). The solid thus obtained was combined with filtration to give the title compound (5.09 g), 1 H NMR (DMSO-d ₆ ) 3.04 (1H), 3.40 (1H), 3.73 (2H), 5 , 18 (1H), 5.29 (1H), 5.73 (1H), 6.21 (1H), 7.19 (2H), 7.81 (1H).

Example 2

Chiral HPLC separation of (±) - (cis) -4-amino-1- (2-hydroxymethyl-L3oxathiolan-5-yl) - (1H) -pyrimidin-2-one enantiomers (a) The racemic product of Example 1 (25 mg ) undergo preparative HPLC under the following conditions:

Column: Merck Hibar cellulose triacetate, 250 x 10 mm, 10 μ

Eluent: ethanol;

Flow rate: 3 ml / min

Detection: uv, 270 nm;

Temperature: normal

Evaporation of the corresponding pooled fractions gave (2R, cis) -4-amino-1- (2-hydroxymethyl, 3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one (6.8 mg, ie about 100 %) and (2S, cis) -4-aminol- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one (3.6 mg, ie about 90%).

(B) The racemic product of Example 1 (26 mg) was subjected to preparative HPLC under the following conditions:

Column: ASTEC cyclobond I acetyl, 250 x 4.6 mm;

Eluent: 0.2% triethylammonium acetate (prepared by adding glacial acetic acid to 0.2% triethylamine in water, to a final pH of 7.2);

Flow rate: 2 ml / min

Detection: uv, 300 nm;

Temperature: normal

Evaporation of the appropriate fractions gave crude (2R, cis) -4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one (25 mg) and crude (2S, cis) -4-amino-1- (2hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one (17 mg). These fractions are separately subjected to further preparative HPLC under the following conditions:

Column: ASTEC cyclobond I acetyl, 250 x 4.6 mm;

Eluent: 15 mM ammonium acetate, pH 6.8);

Flow rate: 0.5 ml / min

Detection: uv, 300 nm;

Temperature: 5 ° C.

V

Evaporation of the corresponding pooled fractions gave (2R, cis) -4-amino-1- (2-hydroxymethyl, 3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one (5.0 mg, ca. 91 %) and (2S, cis) -4-aminol- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one (7.6 mg, ie about 96%).

Example 3 (-) - cis-4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one (i) (±) -cis-4- amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidinone, dihydrogen phosphate, ammonium salt

Cold (0 °), stirred suspension of (±) -cis-4-amino-1- (2-hydroxymethyl-1,3-oxathiolan5-yl) - (1H) -pyrimidin-2-one (1.00 g) ( Example 1) In dry trimethyl phosphate (20 ml) was treated with phosphorus oxychloride (2.44 ml) and the mixture was stirred at 0 ° C for 35 minutes and then quenched in ice water (60 g). The cold mixture was adjusted to pH 2.5 with the addition of aqueous N-sodium hydroxide, then applied to a charcoal column (10 g, DARCO), eluted with water, then with ethanol-aqueous ammonia. The fractions containing crude monophosphate were combined and concentrated. The resulting solution was applied to a column containing 25 g of DEAE Sephadex A-25 (HCO ₅ form). Elution was carried out with a water gradient (120 ml) to 0.1 M-NH 2 HCOj (240 ml), then with 0.2, 0.3 and 4 M NH 2 OHCO 2 (240 ml) (120, 240, or 400 ml). . The relevant fractions are combined and concentrated. The remaining solution was diluted with water (40 ml) and lyophilized to give the title product as a white solid (1.37 g); 7max. (pH 6 buffer), 271.0 nm (E ^7% ; cm 190); ^; H NMR (D 2 O) δ 3.23 (1H), 3.55 (1H), 4.0-4.2 (2H), 5.43 (1H), 6.07 (1H), 6.33 (1H) ), 8.08 (1H).

(ii) (2R, cis) -4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one and (2S, cis) -4-amino -1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one

5'-nucleotidase (snake crotalus atrox) [EC 3.1.3.5] (60 mg at 17 units / mg) was added to a solution of (±) -cis-4-amino- (2-hydroxymethyl-1,3-oxathiolan- 5-yl) (1H) -pyrimidin-2-one, 6'-dihydrogen phosphate, ammonium salt (1.35 g) in buffer [30 ml, prepared from glycine (526 mg) and magnesium chloride (190 mg) in water ( 100 ml) and the mixture was incubated for 2.5 hours at 37 ° C. More enzyme (20 mg) was added and the incubation was continued for a further 3.5 hours. The resulting mixture was applied to a DEAE Sephadex column

A-25 (HCO ₃ -form). Elution was carried out with water (160 ml), then with 0.1, 0.2, 0.3 and 0.4 M NH ₄ HCO ₃ (with 200 ml each). The corresponding fractions containing the first eluted component were combined and evaporated, the residue was chromatographed on a silica column (60 g, Merck 7734), eluting with chloroform-methanol mixtures. Evaporation of the corresponding methanol-ethyl acetate fractions gave (+) - cis-4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one as a white solid (0 , 30 g) [α] D ²¹ + 137 ° (c 1.04 MeOH); Ή NMR (DMSO) δ 3.04 (1H), 3.40 (1H), 3.73 (2H), 5.18 (1H), 5.29 (1H), 5.73 (1H), 6. 21 (1H), 7.19 (2H), 7.81 (1H).

The appropriate Sephadex column fractions containing the second eluted component were combined and evaporated. The residue was dissolved in water (30 ml), treated with alkaline phosphatase (from Escherichia coli) [EC 3.1.3.1] (1.5 ml per 416 units / ml) then incubated for 1 hour at 37 ° C. The solvent was removed by evaporation and the residue was subjected to column chromatography on silica (60 g, Merck 7734), eluting with chloroform-methanol mixtures. Evaporation of the corresponding methanol-ethyl acetate fractions gave (-) - cis-4-amino-1- (2-hydroxymethyl) -1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one as a white solid substance (0.32 g); [α] D ²¹ -132 ° (c. 1.08, MeOH); Ή NMR (DMSO) δ 3.04 (1H), 3.40 (1H), 3.73 (2H), 5.18 (1H), 5.29 (1H), 5.73 (1H), 6. 21 (1H), 7.19 (2H), 7.81 (1H).

Example 4 (-) - Cis-4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) - (1H) -pyrimidin-2-one (i) Three 50 ml flasks with nutrient broth (Oxoid Ltd) were inoculated with one teaspoonful of Escherichia coli (ATCC 23848) each, scraped from an agar plate. The flasks were incubated overnight at 37 ° C with shaking at 250 rpm and then each flask was used to inoculate 41 CDDs (glutamic acid, 3 g / l; MgSO ₄ , 0.2 g / l: K, SO ₄ , 2 , 5 g / l; NaCl, 2.3 g / l, Na ^ PO ^ Hp, 1.1 g / l, NaH ₂ PO ₄ .2H ₂ O, 0.5 g / l cytidine,

1.2 g / l) in 71 ferments. The cultures were fermented at 750 rpm at 37 ° C with aeration at 4 l / min. After growing for 24 hours, cells were harvested by centrifugation (5000 g, 30 minutes) to give 72 g of wet weight. The cell pellet was resuspended in 300 ml of 20mM Tris HC1 buffer (pH 7.5) and digested with sonication (4 x 45 sec). Cellular wastes were determined by centrifugation (30,000 g, 30 minutes) and the protein in the supernatant precipitated by adding ammonium sulfate to 75% saturation. The precipitate was collected by centrifugation (30,000 g, 30 minutes) and resuspended in 25 ml of HEPES buffer (100 mM, pH 7.0) containing ammonium sulfate (75% saturation). The enzyme solution was prepared by centrifugation at 12,000 rpm for 30 minutes.

-

The supernatant was discarded and the pellet was dissolved in Tris HC1 buffer (pH 7.0; 100 mM) to its original volume.

(ii) The product of Example 1 (115 mg) was dissolved in water (100 ml) and stirred. An enzyme solution (0.5 ml) was added and the mixture was maintained at constant pH by continuous addition of HCl (25 mM). The conversion was monitored by chiral HPLC, which showed that the (+) substrate enantiomer was preferentially deaminated. After 25 hours, the (+) enantiomer of the substrate (RT 12.5 min) was completely removed and the solution was adjusted to pH 10.5 with the addition of concentrated sodium hydroxide.

The solution produced above was eluted through a Sephadex QAE column (A25; Pharmacia; 30Χ 1.6 cm) pre-equilibrated to pH 11). The column was washed with water (200 ml) and then with HCl (0.1 M). Fractions (40 ml) were collected and analyzed by reverse phase HPLC. Fractions 5-13 containing the unreacted (-) enantiomer of the substrate were combined and adjusted to pH 7.5 with HCl. The fraction 47 containing the deaminated product was adjusted to pH 7.5 with dilute NaOH. Analysis by chiral HPLC showed that this material was a mixture consisting of one enantiomer (RT

10.2 min) as the major component, with the second enantiomer (RT 8.5 min) as the minority component (about 90%).

(iii) Repeat the above phase (ii) on a larger scale. The compound of Example 1 (363 mg) was incubated in 250 ml of water with enzyme solution (0.5 ml) prepared as in step (i). After 18 and 47 hours, further aliquots (0.5 ml) of the enzyme were added. The reaction mixture was stirred for 70 hours, then allowed to stand for a further 64 hours. Analysis by chiral HPLC showed that the (+) enantiomer of the substrate was completely deaminated, and the resulting solution was adjusted to pH 10.5 with NaOH.

The above solution was filled onto the same QAE column and eluted as in step (i), collecting fractions 2-6 containing a mixture of residual substrate and deaminated product. Fractions 7-13 containing the residual substrate ((-) enantiomer) were collected and adjusted to pH 7.5. Fractions 25-26 containing the deaminated product were collected and neutralized.

The above fractions 2-6 were again eluted through the same QAE column. Fractions 3-11 from this second column contained the unreacted substrate ((-) enantiomer). Fraction 70 contained a deaminated product.

(iv) The separated fractions of the substrate from phases (ii) and (iii) were combined and adjusted to pH 7.5. This solution was eluted through a column of XAD-16 (40x2.4 cm) packed in water. The column was washed with water and then eluted with acetone: water (1: 4 v / v). Fractions containing the (-) enantiomer BCH 189 were collected and lyophilized to give a white powder (190 mg).

The HPLC procedures described above were as follows:

1. Reverse-phase HPLC analysis

Column: Capital Cartridge

Eluent Flow Detection Retention times

Spherisorb ODS-2 (5 μτη)

150 x 4.6 mm ammonium dihydrogen phosphate (50 mM) + 5% MeCN

1.5 ml / min UV, 270 nm

BCH 189 5.5 min; deaminated

BCH -189 8.1 min.

2. Chiral HPLC analysis

Column

Eluent

Flow rate:

Detection

Cyclobond I acetyl 250 x 4.6 mm 0.2% triethylammonium acetate (pH 7.2) 1.0 ml / min

UV, 270 nm

Retention times: BCH 18911.0 and 12.5 min deaminated 189 8.5 and 10.2 min.

(Bioconversion is monitored by registering peak loss at 12.5 min and accumulated product at 10.2 min).

Example 5 (±) -Cis-4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) -phenyl) -pyrimidin-2-one

A full teaspoon of E. coli B cells (ATCC 32848) scraped from a culture plate with nutrient agar is used to inoculate two Florence flasks, each containing 250 ml of nutrient broth. The culture was incubated at 37 ° C with shaking (250

rpm, 5 cm swing) for 18 hours. This is then used to inoculate 40 1 CDDs with cytidine in a 701 fermenter.

The fermentation conditions were as follows: 40 1 / min aeration, stirring speed 750 rpm, at 37 ° C. We install three Rushton propellers in the fermenter. Fermentation was conducted 18 hours before harvest using Sharples continuous centrifuge. The cell paste (150 g net weight) was frozen at -20 ° C before the cells were broken down.

CDD basis g / 1

L-glutamic acid 3

MgSO, 0.2

K ₂ SO ₄ 2.5

NaCl 2,3

On ₂ HPO ₄ 1.1

NaH ₂ PO ₄ 0.6

Prepare it with distilled water. Sterilize at 121 ° C for 30 minutes. Cytidine (1.2 g / l) was sterilized on a filter and added before inoculation.

The frozen cell paste (150 g) was thawed and suspended in 750 ml of 100 mM Hepes (N1 [2-hydroxyethyl] piperazine-N '- [2-ethanesulfonic acid) buffer (pH 7.5) containing 1 mM ethylenediaminetetraacetic acid (sodium salts) and 1 mM dithiothreitol (breakdown buffer). Cells are broken by passing the suspension through a Manton-Gaulin homogenizer operating at 51712.5 kPa. This is done three times by cooling the suspension to about 5 ° C after each passage through the homogenizer. The homogenate was clarified by centrifugation (1400 g; 60 min). Cytidine deaminase activity was adsorbed on a Q-Sepharose column (490 mg bed volume) pre-equilibrated with 50 mM Tris (hydroxymethyl) methylamine (pH 7.5) containing 1 mM sodium chloride. The collected active fractions (210 ml) were collected on a phenyl-Sepharose column (490 ml layer volume) pre-equilibrated with a buffer sample containing 3.2 M ammonium sulfate. The bound enzyme is eluted by a gradient by reducing the concentration of ammonium sulfate. Fractions containing cytidine deaminase activity were collected (695 ml) and the partially purified enzyme was then precipitated with 80% ammonium sulfate. After centrifugation (1400 g; 60 min), the pellet was resuspended in 54 ml of supernatant and stored at 4 ° C.

6.2 ml of this solution were centrifuged (18,000 g, 90 min) and the pill was dissolved in 24 ml of 0.5M potassium phosphate buffer (pH 7.5). The suspension was dialyzed overnight against IM potassium phosphate buffer (pH 7.5). The retentate (20 ml) was then diluted with an equal volume of distilled water. Eupergit C beads (1 g) were added to 35 ml of this solution and the mixture was allowed to stand at room temperature for 150-300 hours (determined by measuring the residual cytidine deaminase activity in the solution). The immobilized enzyme was washed with 100 mM Tris / HCl buffer (pH 7.0) containing 1 mM EDTA, 1 mM DTT, 0.5M NaCl and 500 ppm p-hydroxybenzoic acid ethyl ester (storage buffer). The immobilized enzyme (2.7 g, wet weight) was stored in this buffer at 4 ° C until needed for biotransformation.

The product of Example 1 (3 g) was dissolved in 500 ml of distilled water in a magnetic stirring flask. Bioconversion is carried out at 32 ° C in a pH stat. The pH was maintained constant at 7.0 by the addition of IM acetic acid. Wash 1 g of wet pellet with immobilized enzyme before starting the reaction with distilled water. The conversion was monitored by chiral HPLC, which showed that the (+) enantiomer of the substrate was preferentially deaminated. At the end of the reaction (72 hours), the enzyme beads were filtered and the filtrate was used to isolate the desired (-) enantiomer.

The pH of the reaction mixture was adjusted to pH 10.5 using ammonia solution (IM) and the solution was applied to a Duolite A113 super-resin in a CH cycle (50 ml); 0.4 layer volume per hour). The resin adsorbed analog uridine and the (-) enantiomer pass straight through the column. Any (-) enantiomer still remaining on the resin is removed by washing with 0.04% ammonia solution (2 volume layers; 0.8 flow rate / hour).

The pH of the solutions and liquids washed were washed (600 ml) to pH 7.0 with concentrated sulfuric acid and the solution applied to XAD16 resin (50 ml); flow rate of 1.4 volume layers per hour. The column was washed with distilled water (2.5 volume layers, flow rate 2 volume layers per hour) and the (-) enantiomer was eluted with a 1: 3 mixture of acetomvod (flow rate 1.5 volume layers per hour).

The fraction containing the mass (-) of the enantiomer (4 volume layers) was concentrated on a small volume Buchi evaporator before filtration through glass sinter no. 3. The sintered solution was lyophilized to give 1.2 g of the title product identical to that obtained in Example 4.

Example 6

The tablet formulation

A. The following formulation is prepared by wet granulation of the ingredients with a solution of providone in water, drying and sieving and then adding magnesium stearate and pressing.

mg / tablet (a) Active ingredient 250 (b) Lactose B.P. 210 (c) providon B.P. 15 (d) Sodium starch glycolate 20 (e) Magnesium stearate 5

500

B. The following formulation is prepared by direct compression, with lactose as direct compression.

mg / tablet active ingredient 250 lactose 145 avicel 100 magnesium stearate 5

500

C. (Controlled release formulation). The formulation is prepared by wet granulation of the ingredients (below) with a solution of providone in water, drying and sieving, and then adding magnesium stearate and pressing.

mg / tablet (a) active ingredient 500 (b) hydroxypropylmethylcellulose 112 (Methocel K4M Premium) (c) lactose B.P. 53

(d) providon B.P. 28 (e) magnesium stearate 7

700

Example 7

Capsule formulation

The capsule formulation is performed by mixing the ingredients below and filling them into a two-part hard gelatin capsule.

Active ingredient mg / capsule 125 lactose 72.5 avicel 50 magnesium stearate 2.5 250

Example 8

Formulation for injections

Active ingredient 0,200 g

Sodium hydroxide solution, 0, 1M q.s. to a pH of about 11.

Sterile water q.s. up to 10 ml

The active ingredient is suspended in some water (which can be heated) and the pH is adjusted to about 11 with sodium hydroxide solution. The batch was then filled to the required volume and filtered through a filter with a sterilizing quality membrane into a 10 ml sterile glass vial and closed with a sterile stopper and sealed.

Example 9

The active ingredient is hard fat, B.P mg / suppository

250

1770

2020. - - Ί

Melt one fifth of the fat in a steaming pan at a maximum of 45 ° C. The active ingredient was siphoned through a 200 µm sieve and added to the molten base by stirring using a high shear mixer until a smooth dispersion was obtained. While maintaining the mixture at 45 ° C, the remaining solid fat was added to the suspension and stirred to obtain a homogeneous mixture. The whole suspension was passed through a 250 µm stainless steel sieve and allowed to cool to 40 ° C with continuous stirring. At a temperature of 38 ° C to 40 ° C, 2.02 g of the mixture is filled into suitable 2 ml plastic molds. Allow the candles to cool to room temperature.

Example 10

Biological activity

Antivirus activity

The antiviral activity of the compound of Example 2 was determined against three strains of HIV-1 and one strain of HIV-2 in the following cell lines.

JM cells, a semi-mature T-cell line derived from a patient with HIV-1 strain GB / 3 lymphoblastic leukemia.

C8166 cells, human T-lymphoblastoid cell line, infected with HIV-1 strain RF.

MT-4 cells, a human T-cell human leukemia cell line infected with HIV-1 strain RF.

CEM cells, a human T lymphoblastoid cell line infected with HIV-1 strain RF and U455 or HIV-2 strain ROD.

Antiviral activities in C8166, JM, and CEM cells were determined by inhibiting syncytium formation (Tochikura et al Virology, 164, 542-546) and in MT-4 cells by inhibiting formazan conversion (Baba et al; (1987) Biochem. Biophys. Res. Commun. 142 128134; Mossman (1983) J. Immun. Meth .; 65, 55-57). Antiviral activities are also determined by analyzing the amount of HIV p24 antigen synthesized in the presence and absence of the enantiomer.

The results are shown in Tables 1 and 2 below:

j ». r \ r ·;

j, · «* • 'ΐ • Λ

V * Λ · 'ίί

Table 1

50% antiviral activity (^ µg / ml) . Test Form zan Inhibition of syncytium formation Cells MT-4 CEM CEM CEM JM C8166 Virus (H (V ”1) HIV-1 RF • HIV-2-ROD HIV-1 RF HIV-1 U455 HIV-1GB8 HIV-1 RF {+) - Enantiomer 0.28 0.045 0.07 0.006 0.03 0.05 (-) - Enantiomer 0.20 o rčrss 0.04 0.008 0.05 0.01

Table 2

Inhibition of HIV p24 synthesis

50% inhibition of HIV p24 synthesis (^ µg / ml) Cells C8166 JM MT-4 Virus RF GB8 RF (+) - Enantiomer 0.021 0.033 0.0008 (-) - Enantiomer 0.016 0.016 0.0004

B. Cytotoxicity

The cytotoxicity of the compounds of Example 2, racemic compound (BCH-189; Example 1) and 50/50 mixtures of the two enantiomers was determined in five CD4 cell lines: H9, JM, CEM, C8166 and U937.

The test compounds were serially diluted from 100 + g / ml to 0.3 gg / ml (final concentrations) in 96-well microtiter plates. 3.6 χ 10 ⁴ cells were incubated in each well, including drug-free controls. After incubation for 5 days at 37 ° C, viable cell counts were performed by removing the cell suspension sample and counting in a hemocytometer with Trypan Blue cells excluded.

Table 3

Cytotoxicity

50Χ Cytotoxicity of CpgSj &} Compound CEN cel. JM cel. K9 чел . U937 forehead C8186 чел. C ♦) -enantiomer 1 < 1.5 2 4 35 C -> - Enantiomer > 150 30 > 100 > 100 > 100 BCH-1A8S 3 3.5 8 15 SO 1: 1 mixture 2.5 ND * ND ND ND

ND »not specified

For

BioChem Pharma Inc .:

v η fi- ''

Claims (16)

Patent claims A process for the preparation of (-) - 4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) (1H) -pyrimidin-2-one or a pharmaceutically acceptable derivative thereof (Compound A) by separating the (-) - enantiomer from the mixture, which also contains the (+) - enantiomer. Process according to claim 1, characterized in that compound (A) is obtained essentially without the corresponding (-f -) - enantiomer. Process according to claim 1 or claim 2, characterized in that in the compound (A) (+) - enantiomer thus obtained is not present in an amount greater than 5% w / w. Process according to any one of claims 1 to 3, characterized in that the (A) (+) - enantiomer present in the compound thus obtained is present in an amount of not more than about 2% w / w. Process according to any one of claims 1 to 4, characterized in that in the compound (A) (+) - enantiomer thus obtained is present in an amount less than about 1% w / w. Process according to claim 1, characterized in that compound (A) is obtained in substantially pure form. Process according to any one of claims 1 to 6, characterized in that it comprises separating the (-) - enantiomer from a mixture also containing (+) - the enantiomer. A process according to any one of claims 1 to 7, characterized in that the mixture of compounds is in the racemic mixture. A method according to any one of claims 1 to 8, characterized in that the separation is carried out by chiral HPLC. 10. The method of claim 9, wherein the HPLC uses, as a stationary phase, acetylated / 3-cyclodextrin or cellulose triacetate. A method according to any one of claims 1 to 8, characterized in that the separation is carried out by enzymatically mediated enantioselective catabolism. A method according to claim 11, characterized in that the enzyme is used in immobilized form. The method of claim 11 or claim 12, wherein the enzyme is cytidine deaminase. A method according to claim 11 or claim 12, characterized in that the enzyme is 5′-nucleotidase. Use of a compound prepared by the method of any one of claims 1 to 14 for the manufacture of a medicament for treating a viral infection. A process for the preparation of a pharmaceutical formulation comprising as an active ingredient a compound as prepared by the process as defined in any one of claims 1 to 14, together with a pharmaceutically acceptable carrier therefor, characterized in that it comprises mixing the active ingredient and the carrier .